This research program is designed to test the efficacy of candidate drugs, for protection against inner ear receptor cell (hair cell) loss and hearing loss resulting from systemic treatment of patients with gram- negative antibiotics. Loss of inner ear hair cells is the leading cause of hearing loss and balance dysfunction, affecting approximately 31 million Americans. The candidate drugs under investigation will be analogs of PR0T01, a small molecule, drug-like compound discovered using a well-characterized chemical screen of lateral line mechanosensory hair cells of larval zebrafish, in vivo. PR0T01 exposure provides robust protection of zebrafish hair cells hair cells against a broad range of aminoglycoside antibiotic conditions and exposure levels, and protects rodent inner ear hair cells in vitro and in vivo. The following Specific aims are proposed. 1) Screen analogs of PR0T01 for those hits with optimized protection of hair cells and other drug characteristics using a well characterized, robust and sensitive zebrafish lateral line hair cell assay. 2) Evaluate the most promising new PROTO analogs with secondary assays to determine lead compounds from among promising hits, based on detailed assessment of neomycin-induced hair cell loss, acute and chronic gentamicin-induced hair cell loss, normal efficacy of aminoglycoside as a bacteriostatic agent via MIC/MBL tests, and uptake of the aminoglycoside by hair cells. These experiments will continue to utilize the larval zebrafish hair cell platform. 3) Evaluate lead PROTO analogs for their ability to confer hearing protection against aminoglycoside-induced damage in vivo in mature rats by testing with a well-established auditory brain stem response (AER) assay and by quantitative assessment of inner ear hair cell integrity, 4) Direct IND and phase I clinical preparation of lead compounds in conjunction with NIH supported contractors. The studies outlined in the specific aims will provide a focused analysis of the ability of PR0T01 analogs to protect hair cells from drug-induced damage. Optimization of lead compounds and validation in a mammalian system will provide essential drug development information for future clinical trials in human patients.